Increased Expression of YAP Inhibited the Autophagy Level by Upregulating mTOR Signal in the Eutopic ESCs of Endometriosis

Abstract

We first reported that the Hippo-YAP signaling pathway plays a critical role in the pathogenesis of endometriosis (EMS). Autophagy is also related to the invasion ability of endometrial cells and is involved in the pathogenesis of EMS through multi-levels. However, the precise regulatory mechanism of YAP on autophagy in the eutopic endometrial stromal cells (ESCs) is still unclear. Primary eutopic ESCs of EMS patients (n = 12) and control patients without EMS (n = 9) were isolated and cultured to investigate the expressions of YAP and mTOR, the role of YAP in autophagy, and the effect of the YAP-autophagy signal on the decidualization of the eutopic ESCs. Endometriosis-related sequencing data (GSE51981) in the GEO database were used to find the genes significantly correlated with YAP. We found 155 genes with significant differences in the interaction with YAP in EMS from the dataset, and the autophagy pathway was significantly enriched. Following on from our previous studies of YAP knockdown, overexpression of YAP resulted in an increased expression of mTOR and decreased ratio of LC3-II/LC3-I and autophagy markers, in the eutopic ESCs; transmission electron microscope observation also showed fewer autophagosomes compared with the control cells. Furthermore, ESCs of the Rapamycin-treated group showed significant decidual-like changes with significantly increased decidual prolactin level at 72 h after in vitro decidualization. These results demonstrate that the increased YAP inhibited the level of autophagy by upregulating the mTOR signal in the eutopic ESCs of endometriosis. The YAP-autophagy signal plays an important role in the pathogenesis of endometriosis-associated infertility.

Keywords: Yes-associated protein (YAP); autophagy; decidualization; endometriosis; eutopic endometrial stromal cells (eutopic ESCs); mammalian target of rapamycin (mTOR).

Figure 1

Volcano Plot and PPI network of DEGs. In the Volcano Plot, genes that are upregulated are in red, those that are downregulated are in blue, and those that are insignificantly different are in black (A). Different protein interaction graph with YAP interaction scores greater than 0.4 in GSE51981 samples were shown in the PPI network. Green indicates downregulated expression and red indicates upregulated expression (B).

Figure 2

GO and KEGG enrichment analysis of the 155 genes significantly associated with YAP. (A) The abscissa is the percentage of genes under the GO functional module (ratio); the ordinate is the GO functional module: BP (biological process), CC (cell composition), and MF (molecular function). (B) The x-coordinate in the figure is the number of DEGs annotated to KEGG pathway/the total number of differentially expressed genes (ratio); the ordinate is the KEGG pathway. The size of the point represents the number of DEGs annotated to the KEGG pathway.

Figure 3

The protein locations of YAP and mTOR in the eutopic ESCs. The results of immunofluorescence showed that YAP was mainly expressed in the nucleus of the eutopic ESCs and a little in the cytoplasm, whereas in the normal ESCs, YAP was mainly located in the cytoplasm and slightly expressed in the nucleus. mTOR was mainly expressed in the cytoplasm of the eutopic ESCs and a little in the nucleus, whereas mTOR was expressed in small amounts in the cytoplasm of normal ESCs, but it was hardly expressed in the nucleus.

Figure 4

Overexpression of YAP in the eutopic ESCs of endometriosis inhibited autophagy level. The qPCR showed that the expression of YAP mRNA was increased significantly (3.60 ± 0.16 vs. 1.00 ± 0.21; P = 0.0006) after transfection with YAP-overexpression plasmid in the eutopic ESCs compared with controls (the eutopic ESCs transfected with the empty plasmid) (A). Western blotting revealed that we obtained a high OE efficiency at the YAP protein level (3.04 vs. 1.00) (B). The expression of mTOR protein (2.11 vs. 1.00) was significantly increased in the eutopic ESCs compared with controls following YAP-OE. By contrast, there was a significantly decreased ratio of the autophagy marker protein LC3-II/LC3-I (0.44 vs. 1.00) (C). ***p = 0.0006.

Figure 5

Transmission electron microscope (TEM) observation of autophagosomes after overexpression of YAP in the eutopic ESCs of endometriosis. TEM observation showed the fewer autophagosomes in the YAP-OE group compared with the control cells.

Figure 6

Effects of Verteporfin and Rapamycin treatments on the in vitro decidualization of ESCs. Compared to the cultured control group, the morphology of ESCs in the Verteporfin treatment group had no obvious decidual changes after 24 h, 48 h, and 72 h of in vitro induction (A). ELISA results suggested that there was no significant difference in the dPRL levels in the culture medium of ESCs in the groups of Verteporfin and the control group after 24 h, 48 h, and 72 h of decidualization (p > 0.05) (B). After 24 h and 48 h of in vitro induction, some of the ESCs in the Rapamycin-treated and control groups were rounded and enlarged. Seventy-two hours after in vitro induction, compared with the control group, the Rapamycin-treated ESCs showed obvious decidual-like changes (C). After 72 h of decidualization, the dPRL level was significantly increased in the cell culture medium of the Rapamycin group compared with that of the control group (p < 0.05) (D). *p < 0.05.